Clotrimazole causes membrane depolarization and induces sub G0 cell cycle arrest in Leishmania donovani.

Clotrimazole is an FDA approved drug and is widely used as an antifungal agent. An extensive body of research is available about its mechanism of action on various cell types but its mode of killing of Leishmania donovani parasites is unknown. L. donovani causes Visceral Leishmaniasis which is a public health problem with limited treatment options. Its present chemotherapy is expensive, has adverse effects and is plagued with drug resistance issues. In this study we have explored the possibility of repurposing clotrimazole as an antileishmanial drug. We have assessed its efficacy on the parasites and attempted to understand its mode of action. We found that it has a half-maximal inhibitory concentration (IC50) of 35.75 ± 1.06 µM, 12.75 ± 0.35 µM and 73 ± 1.41 µM in promastigotes, intracellular amastigotes and macrophages, respectively. Clotrimazole is 5.73 times more selective for the intracellular amastigotes as compared to the mammalian cell. Effect of clotrimazole was reduced by ergosterol supplementation. It leads to impaired parasite morphology. It alters plasma membrane permeability and disrupts plasma membrane potential. Mitochondrial function is compromised as is evident from increased ROS generation, depolarized mitochondrial membrane and decreased ATP levels. Cell cycle analysis of clotrimazole treated parasites shows arrest at sub-G0 phase suggesting apoptotic mode of cell death.

Upregulation of ATP6V0D2 benefits intracellular survival of Leishmania donovani in erythrocytes-engulfing macrophages.

Visceral leishmaniasis (VL) is the most severe type of leishmaniasis which is caused by infection of Leishmania donovani complex. In the BALB/c mouse model of VL, multinucleated giant cells (MGCs) with heavy parasite infection consist of the largest population of hemophagocytes in the spleen of L. donovani-infected mice, indicating that MGCs provide the parasites a circumstance beneficial for their survival. Although ATP6V0D2 is a demonstrated factor inducing the formation of hemophagocytic MGCs during L. donovani infection, functions of this protein in shaping the infection outcome in macrophages remain unclear. Here we evaluated the influence of upregulated ATP6V0D2 on intracellular survival of the parasites. L. donovani infection-induced hemophagocytosis of normal erythrocytes by macrophages was suppressed by RNAi-based knockdown of Atp6v0d2. The knockdown of Atp6v0d2 did not improve the survival of amastigotes within macrophages when the cells were cultured in the absence of erythrocytes. On the other hand, reduced intracellular survival of amastigotes in macrophages by the knockdown was observed when macrophages were supplemented with antibody-opsonized erythrocytes before infection. There, increase in cytosolic labile iron pool was observed in the L. donovani-infected knocked-down macrophages. It suggests that ATP6V0D2 plays roles not only in upregulation of hemophagocytosis but also in iron trafficking within L. donovani-infected macrophages. Superior access to iron in macrophages may be how the upregulated expression of the molecule brings benefit to Leishmania for their intracellular survival in the presence of erythrocytes.

Swarna Bhasma Induces Antigen-Presenting Abilities of Macrophages and Helps Antigen Experienced CD4+ T Cells to Acquire Th1 Phenotypes Against Leishmania donovani Antigens.

In leishmaniasis, the protective immunity is largely mediated by proinflammatory cytokine producing abilities of T cells and an efficient parasite killing by phagocytic cells. Notwithstanding a substantial progress that has been made during last decades, the mechanisms or factors involved in establishing protective immunity against Leishmania are not identified. In ancient Indian literature, metallic "bhasma," particularly that of "swarna" or gold (fine gold particles), is indicated as one of the most prominent metal-based therapeutic medicine, which is known to impart protective and curative properties in various health issues. In this work, we elucidated the potential of swarna bhasma (SB) on the effector properties of phagocytes and antigen-activated CD4+ T cells in augmenting the immunogenicity of L. donovani antigens. The characterization of SB revealing its shape, size, composition, and measurement of cytotoxicity established the physiochemical potential for its utilization as an immunomodulator. The activation of macrophages with SB enhanced their capacity to produce nitric oxide and proinflammatory cytokines, which eventually resulted in reduced uptake of parasites and their proliferation in infected cells. Further, in Leishmania-infected animals, SB administration reduced the generation of IL-10, an anti-inflammatory cytokine, and enhanced pro-inflammatory cytokine generation by antigen activated CD4+ T cells with increased frequency of double (IFNγ+/TNFα+) and triple (IFNγ+TNFα+IL-2+) positive cells and abrogated disease pathogeneses at the early days of infection. Our results also suggested that cow-ghee (A2) emulsified preparation of SB, either alone or with yashtimadhu, a known natural immune modulator which enhances the SB's potential in enhancing the immunogenicity of parasitic antigens. These findings suggested a definite potential of SB in enhancing the effector functions of phagocytes and CD4+ T cells against L. donovani antigens. Therefore, more studies are needed to elucidate the mechanistic details of SB and its potential in enhancing vaccine-induced immunity.

Screening of traditional medicinal plant extracts and compounds identifies a potent anti-leishmanial diarylheptanoid from Siphonochilus aethiopicus.

Available anti-leishmanial drugs are associated with toxic side effects, necessitating the search for safe and effective alternatives. This study is focused on identifying traditional medicinal plant natural products for anti-leishmanial potential and possible mechanism of action. Compounds S and T. cordifolia residual fraction (TC-5) presented the best anti-leishmanial activity (IC50: 0.446 and 1.028 mg/ml) against promastigotes at 48 h and less cytotoxicity to THP-1 macrophages. These test agents elicited increased expression of pro-inflammatory cytokines; TNFα and IL-12. In infected untreated macrophages, NO release was suppressed but was significantly (p < 0.05) increased in infected cells treated with compound S. Importantly, Compound S was found to interact with LdTopoIIdimer in silico, resulting in a likely reduced ability of nucleic acid (dsDNA)-remodelling and, as a result, parasite proliferation in vitro. Thereby, Compound S possesses anti-leishmanial activity and this effect occurs via a Th1-mediated pro-inflammatory response. An increase in NO release and its inhibitory effect on LdTopoII may also contribute to the anti-leishmanial effect of compound S. These results show the potential of this compound as a potential starting point for the discovery of novel anti-leishmanial leads.Communicated by Ramaswamy H. Sarma.

TLR-2 agonist Pam3CSK4 has no therapeutic effect on visceral leishmaniasis in BALB/c mice and may enhance the pathogenesis of the disease.

Most of the existing Leishmania-related research about TLR-2 agonists was focusing on their role as adjuvants in the vaccine, few studied its therapeutic effect. This paper aims to explore the therapeutic effect of TLR-2 agonist Pam3CSK4 on Leishmania-infected mice and the underlying immune molecular mechanisms. In L. donovani-infected BALB/c mice, one group was treated with Pam3CSK4 after infection and the other group was not treated. Normal uninfected mice treated with Pam3CSK4 or untreated were used as controls. Parasite load, hepatic pathology and serum antibodies were detected to assess the severity of the infection. The expression of immune-related genes, spleen lymphocyte subsets and liver RNA-seq were employed to reveal possible molecular mechanisms. The results showed that the liver and spleen parasite load of infected mice in Pam3CSK4 treated and untreated groups had no statistical difference, indicating Pam3CSK4 might have no therapeutic effect on visceral leishmaniasis. Infected mice treated with Pam3CSK4 possessed more hepatic inflammation focus, lower IgG and IgG2a antibody titers, and a lower proportion of spleen CD3+CD4+ T cells. Transcriptome analysis revealed that Th1/Th2 differentiation, NK cells, Th17 cell, complement system and calcium signaling pathways were down-regulated post-treatment of Pam3CSK4. In this study, TLR-2 agonist Pam3CSK4 showed no therapeutic effect on visceral leishmaniasis in BALB/c mice and might enhance the pathogenesis of the disease possibly due to the down-regulation of several immune-related pathways, which can improve our understanding of the role of TLR-2 in both treatment and vaccine development.

New antiprotozoal sesquiterpene derivatives from Dorema glabrum Fisch & C.A.Mey.

Seven undescribed sesquiterpene derivatives, Azerins A-G (3-6, 8, 14 and 15), three known sesquiterpene phenols, kopetdaghin A (1), kopetdaghin B (2) and latisectin (7), together with five known sesquiterpene coumarins (9-13), were isolated from the roots of Dorema glabrum. The structures were elucidated by comprehensive 1D- and 2D-NMR spectral analysis as well as HR-ESI-MS. Compounds were assessed for their in vitro antiprotozoal activity against Trypanosoma brucei rhodesiense, T. cruzi, Leishmania donovani, and Plasmodium falciparum. Cytotoxic potentials of the compounds were also tested on L6 rat skeletal myoblasts. Azerin G (15) showed a potent preferential growth inhibitory activity against T. b. rhodesiense with IC50 value of 0.01 µM and selectivity index of 329. Compounds 1, 4, 7 and 8 were also found as the most active compounds with selective growth inhibitory effects toward P. falciparum with selectivity indices ranging from 11.6 to 16.7 (IC50: 1.8-24.6 µM).

Andrographolide-Soya-L-α-Phosphatidyl Choline Complex Augmented Solubility and Drug Delivery in Leishmania donovani, a Causative Agent for Cutaneous and Visceral Leishmaniasis.

The utility of andrographolide (AN) in visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL) is limited owing to poor solubility, hindered permeation, and unstable structure under physiological conditions. The present study mainly focuses on synthesizing of andrographolide-Soya-L-α-phosphatidyl choline (ANSPC) complex in ethanol and its characterization using various spectral and analytical techniques. Results from FT-IR, 1H NMR, ROSEY, and in silico docking techniques suggest ANSPC complex formation due to inter-molecular interaction between the hydrophilic head of SPC and hydroxyl group of AN present at 24th position. ANSPC complex demonstrated the solubility of 113.93 ± 6.66 µg/mL significantly (P < 0.05) greater than 6.39 ± 0.47 µg/mL of AN. The particle size of ANSPC complex was found to be 182.2 ± 2.69 nm. The IC50 value of AN suspension (PBS, pH ~ 7.4) at 24, 48, and 72 h against Leishmania donovani (L. donovani) was noticed to be 32.76 ± 4.53, 20.87 ± 2.37, and 17.71 ± 3.06 µM/mL, respectively. Moreover, augmented aqueous solubility of ANSPC complex led to significant (P < 0.05) reduction in IC50 value, i.e., 25.02 ± 4.35, 11.31 ± 0.60, and 8.33 ± 2.71 µM/mL at 24, 48, and 72 h, respectively. The IC50 values for miltefosine were noted to be 9.84 ± 2.65, 12.13 ± 7.26, and 6.56 ± 0.61 µM/mL at similar time periods. Moreover, ANSPC complex demonstrated augmented cellular uptake at 24 h as compared to 6 h in L. donovani. We suppose that submicron size and phospholipid-mediated complexation might have endorsed the permeation of ANSPC complex across the plasma membrane of L. donovani parasite by transport mechanisms such as P-type ATPase. ANSPC complex warrants further in-depth in vivo studies under a set of stringent parameters for translating the product into a clinically viable form.

Drug repurposing based novel anti-leishmanial drug screening using in-silico and in-vitro approaches.

Visceral leishmaniasis is a neglected tropical disease and is mainly caused by L. donovani in the Indian subcontinent. The mitochondria genome replication in Leishmania spp. is having a very specific mechanism, and it is initiated by a key enzyme called mitochondrial primase. This enzyme is essential for the onset of the replication process and growth of the parasite. Therefore, we focused on the primase protein as a potential therapeutic target for combating leishmaniasis diseases. We started our studies molecular modeling and followed by docking of the FDA-approved drug library into the binding site of the primase protein. The top 30 selected compounds were subjected for molecular dynamics studies. Also, the target protein was cloned, purified, and tested experimentally (primase activity assays and inhibition assays). Some compounds were very effective against the Leishmania cell culture. All these approaches helped us to identify few possible novel anti-leishmanial drugs such as Pioglitazone and Mupirocin. These drugs are effectively involved in inhibiting the promastigote of L. donovani, and it can be utilized in the next level of clinical trials. Communicated by Ramaswamy H. Sarma.

Retinoic Acid Increases Cellular Cholesterol in Leishmania donovani-Infected Macrophages in an mTOR-Independent Manner.

Infection with Leishmania donovani reduces cellular cholesterol and thus deprives the host cells by inhibiting its synthesis and uptake. Changes in cholesterol levels increase the chance of attachment and internalization of L. donovani in macrophages (Mϕ). Retinoic acid (RA), an important micronutrient, restores the lysosomal uptake of cholesterol in L. donovani-infected Mϕ. Importantly, mammalian (or mechanistic) target of rapamycin complex 1 (mTORC1) increases the cellular cholesterol level by increasing expression of sterol regulatory element-binding protein 2 (SREBP2). Whether the efficacy of RA in L. donovani-infected Mϕ is mediated by mTOR is not yet established. Moreover, there are contradicting reports suggesting potential activation and inhibition of mTOR in L. donovani-infected Mϕ. Intrigued by this, we attempted to understand the RA-mediated restoration of cholesterol as well as the possible roles of mTORC1, if any. Our findings suggest that L. donovani infection impairs the synthesis of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR), uptake of low-density lipoprotein receptor (LDLR), and secretion of ATP-binding cassette transporter (ABCA1) in Mϕ. L. donovani infection possibly impairs mTORC1 formation, as it inhibits the expression of regulatory-associated protein of mammalian target of rapamycin (RAPTOR). Importantly, all these are restored upon RA supplementation. RA also restores the levels of SREBP2 in L. donovani-infected Mϕ, resulting in increased cellular cholesterol and thus reducing the parasite burden. When mTORC1 was inhibited, RA exerted a similar response in L. donovani-infected Mϕ; i.e., it restored cholesterol levels and reduced the parasite burden. In summary, RA restores cholesterol levels in L. donovani-infected Mϕ and reduces the parasite burden in an mTOR-independent manner. IMPORTANCE People who reside in regions where leishmaniasis is endemic and who lack proteins, iron, zinc, and vitamin A in their diet are more prone to develop visceral leishmaniasis (VL) as a full-blown disease. Vitamin A deficiency favors the development of a parasitic infection in the human host, and the WHO recommends administering 200,000-IU doses to VL patients on admission. Additionally, Leishmania entry and its survival inside the host are achieved by utilizing host cholesterol, as all trypanosomatids lack de novo synthesis of sterol. We have already shown that RA regulates cellular cholesterol levels associated with an efficient immune response. A deficiency of retinoic acid (RA) favors the parasite in Leishmania donovani-infected macrophages by downregulating the immune response. In the present work, we observed that RA restores cellular cholesterol levels in Leishmania donovani-infected macrophages. This study proposes using RA as an immune potentiator along with standard therapy.

Therapeutic Potential of Quercetin-Loaded Nanoemulsion against Experimental Visceral Leishmaniasis: In Vitro/Ex Vivo Studies and Mechanistic Insights.

Visceral leishmaniasis (VL) is one of the most fatal and neglected tropical diseases caused by Leishmania donovani (L. donovani). The applications of currently available chemotherapy (amphotericin B, miltefosine, and others) in VL treatment have been limited due to their poor bioavailability, unfavorable toxicity profile, and prolonged parenteral dosing. Quercetin (QT), a potent natural antioxidant, is a prominent target when conducting investigations on alternative therapies against L. donovani infections. However, the therapeutic applications of QT have been restricted due to its low solubility and bioavailability. In the present study, we developed and evaluated the antileishmanial activity (ALA) of quercetin-loaded nanoemulsion (QTNE) against L. donovani clinical strains. In vitro anti-promastigote assay results demonstrated that QTNE (IC50 6.6 µM, 48 h) significantly inhibited the growth of parasites more efficiently than the pure QT suspension in a dose- and time-dependent manner. Results of the anti-amastigote assay revealed that the infected macrophages (%) of QTNE were significantly more than those of the pure QT suspension at all concentrations (6.6, 26.4, and 52.8 µM; p < 0.05, p < 0.01 compared to the control). Moreover, the results of in vitro and ex vivo studies assisted in determining the mechanistic insights associated with the ALA of QTNE. The overall findings suggested that QTNE exhibited potential ALA by enhancing the intracellular ROS and nitric oxide levels, inducing distortion of membrane integrity and phosphatidylserine release (AV/PI), rupturing the parasite DNA (late apoptosis/necrosis process), and upregulating the immunomodulatory effects (IFN-γ and IL-10 levels). Additionally, QTNE showed superior biocompatibility against all of the treated healthy cells (PBMCs, PECs, and BMCs) as compared to the control. In conclusion, QTNE acts as a potential antileishmanial agent targeting both promastigote and intracellular amastigote forms of L. donovani, which thus opens a new avenue for the use of QTNE in VL therapy.

Ascaridole exerts the leishmanicidal activity by inhibiting parasite glycolysis.

BACKGROUND: The global burden of leishmaniasis is exacerbated by the limited repertoire of drugs, resulting in an urgent need to develop new therapeutic alternatives. Endoperoxides like ascaridole have emerged as promising anti-parasitic candidates, and its effectiveness was established in an animal model of cutaneous leishmaniasis (CL). However, its impact on Leishmania donovani parasites, causative of visceral leishmaniasis (VL) remains to be established. PURPOSE: This study aimed to delineate the underlying mechanisms contributing towards the leishmanicidal effect of ascaridole in terms of its impact on the cellular redox status and metabolic bioenergetics of L. donovani parasites. METHODOLOGY: The anti-promastigote activity of ascaridole was established by a cell viability assay in L. donovani [MHOM/IN/1983/AG83] and anti-amastigote activity by microscopy and ddPCR (droplet digital polymerase chain reaction). The cellular redox status, mitochondrial membrane potential (MMP), annexin V positivity and cell cycle arrest was evaluated by flow cytometry, while cellular and mitochondrial bioenergetics was assessed using Agilent XFp Analyzer, and the levels of ATP was measured by chemiluminescence. RESULTS: Ascaridole demonstrated strong anti-promastigote and anti-amastigote activities in l. donovani, IC50 (half maximal Inhibitory concentration) being 2.47 ± 0.18 µM and 2.00±0.34 µM respectively, while in J774.A1 and murine peritoneal macrophages, the CC50 (half maximal cytotoxic concentration) was 41.47 ± 4.89 µM and 37.58 ± 5.75 µM respectively. Ascaridole disrupted the redox homeostasis via an enhanced generation of reactive oxygen species (ROS), lipid peroxidation and concomitant depletion of thiols. However, it failed to increase the generation of mitochondrial superoxide, which minimally impacted on mitochondrial respiration and was corroborated by energy metabolism studies. Instead, ascaridole inhibited glycolysis of promastigotes, caused a loss in MMP, which translated into ATP depletion. In promastigotes, ascaridole enhanced annexin-V positivity and caused a cell cycle arrest at sub- G0/G1 phase. CONCLUSION: In summary, ascaridole displays its leishmanicidal activity possibly due to its ability to auto-generate free radicals following cleavage of its endoperoxide bridge that led to disruption of the redox homeostasis, inhibition of glycolysis and culminated in an apoptotic like cell death.

Isolation and Structural Elucidation of Compounds from Pleiocarpa bicarpellata and Their In Vitro Antiprotozoal Activity.

Species of the genus Pleiocarpa are used in traditional medicine against fever and malaria. The present study focuses on the isolation and identification of bioactive compounds from P. bicarpellata extracts, and the evaluation of their antiprotozoal activity. Fractionation and isolation combined to LC-HRMS/MS-based dereplication provided 16 compounds: seven indole alkaloids, four indoline alkaloids, two secoiridoid glycosides, two iridoid glycosides, and one phenolic glucoside. One of the quaternary indole alkaloids (7) and one indoline alkaloid (15) have never been reported before. Their structures were elucidated by analysis of spectroscopic data, including 1D and 2D NMR experiments, UV, IR, and HRESIMS data. The absolute configurations were determined by comparison of the experimental and calculated ECD data. The extracts and isolated compounds were evaluated for their antiprotozoal activity towards Trypanosoma brucei rhodesiense, Trypanosoma cruzi, Leishmania donovani, and Plasmodium falciparum, as well as for their cytotoxicity against rat skeletal myoblast L6 cells. The dichloromethane/methanol (1:1) root extract showed strong activity against P. falciparum (IC50 value of 3.5 µg/mL). Among the compounds isolated, tubotaiwine (13) displayed the most significant antiplasmodial activity with an IC50 value of 8.5 µM and a selectivity index of 23.4. Therefore, P. bicarpallata extract can be considered as a source of indole alkaloids with antiplasmodial activity.

The Potential of 2-Substituted Quinolines as Antileishmanial Drug Candidates.

There is a need for new, cost-effective drugs to treat leishmaniasis. A strategy based on traditional medicine practiced in Bolivia led to the discovery of the 2-substituted quinoline series as a source of molecules with antileishmanial activity and low toxicity. This review documents the development of the series from the first isolated natural compounds through several hundred synthetized molecules to an optimized compound exhibiting an in vitro IC50 value of 0.2 µM against Leishmania donovani, and a selectivity index value of 187, together with in vivo activity on the L. donovani/hamster model. Attempts to establish structure-activity relationships are described, as well as studies that have attempted to determine the mechanism of action. For the latter, it appears that molecules of this series act on multiple targets, possibly including the immune system, which could explain the observed lack of drug resistance after in vitro drug pressure. We also show how nanotechnology strategies could valorize these drugs through adapted formulations and how a mechanistic targeting approach could generate new compounds with increased activity.

Retinoic acid restores the levels of cellular cholesterol in Leishmania donovani infected macrophages by increasing npc1 and npc2 expressions.

Visceral leishmaniasis (VL) is a fatal form among all forms of leishmaniasis and is caused by visceralization of the Leishmania donovani (Ld) parasite to the critical organs. Mild to severe malnutrition is common in VL patients and the deficiency of retinoic acid (RA), an important micronutrient, results in a compromised state of immune response in macrophages (mφ) leading to the increased parasite load. In the continuation of our earlier work, we observed loss of cellular cholesterol in infected mφ in the absence of RA i.e., upon inhibition of RALDH pathway. Moreover, the Leishmania utilizes host cholesterol for the establishment of infection and causes a decrease in the expressions of Niemann-Pick C2 (npc2) and Niemann-Pick C1 (npc1) genes involved in the uptake of extracellular cholesterol. This results in reduced levels of cellular cholesterol in infected mφ. Intrigued by this, as the first sign of our hypothesis, we investigated the presence of RA Response Element (RARE) sequences in the upstream of npc1 and npc2 genes. To functionally confirm this, we measured their expressions and the levels of cellular cholesterol in Ld infected mφ in the absence (i.e., using an inhibitor of RALDH pathway) and presence of RA. We found restoration of the levels of cellular cholesterol in infected mφ under the supplementation of RA resulting in the decreased parasite load. Hence, the supplementation of RA with the standard therapy and/or preventive use of RA could be potentially an advancement in the treatment and cure of VL patients.

Evidence for Reversal of Immunosuppression by Homeopathic Medicine to a Predominant Th1-type Immune Response in BALB/c Mice Infected with Leishmania donovani.

BACKGROUND: Visceral leishmaniasis (VL) is a neglected tropical disease that is fatal if treatment is not given. The available chemotherapeutic options are unsatisfactory, and so complementary therapies like homeopathy might be a promising approach. METHODS: A nosode from a pure axenic culture of Leishmania donovani was prepared and screened for its anti-leishmanial potential both in an in-vitro and an in-vivo experimental approach. RESULTS: Leishmania donovani amastigote promastigote nosode (LdAPN 30C) exhibited significant anti-leishmanial activity against the promastigote forms of Leishmania donovani and was found to be safe. A study conducted on VL-infected mice revealed that LdAPN 30C resolved the disease by modulating the host immune response toward the Th1 type through upregulating the pro-inflammatory cytokines (IFN-γ and IL-17) and inducing nitric oxide (NO) levels in the infected macrophages. The hepatic parasite load was also found to be significantly decreased. The nosode was found to be safe, as no histological alterations in the liver or kidney were observed in the animals treated with the LdAPN 30C. CONCLUSION: This is the first study in which an axenic culture of Leishmania donovani has been used for the preparation of a homeopathic medication. The study highlights the anti-leishmanial and immunomodulatory potential of a homeopathic nosode in experimental VL.

Embilica officinalis L. inhibits the growth and proliferation of Leishmania donovani through the induction of ultrastructural changes, mitochondrial dysfunction, oxidative stress and apoptosis-like cell death.

Visceral leishmaniasis (VL) is caused by a protozoan parasite, Leishmania donovani (L. donovani). It affects around 1-2 million people around the world annually. There is an urgent need to investigate new medicament of it due to difficult method of drug administration, long period of treatment, high cost of the drug, adverse side-effects, low efficacy and development of parasite resistance to the available drugs. Medicinal plants have also been used for the treatment of different diseases in traditional system of medicines due to their holistic effects. The Drugs for Neglected Diseases initiative (DNDi), Geneva, Switzerland has already started the program for identification of potential medicinal plant and plant products having antileishmanial potential. Keeping all these in consideration, we planned to study the antileishmanial activity of one of the medicinal plant, Embilica officinalis L. (EO) fruit extract. EO fruit extract inhibited the growth and proliferation of promastigotes as well as intra-macrophagic amastigotes in dose-dependent manner. EO fruit extract induced morphological and ultrastructural changes in parasites as observed under Electron Microscope. It also induced the oxidative stress, mitochondrial dysfunction, DNA laddering and apotosis-like cell death in parasites. Here, we for the first time reported such a detailed mechanism of action of antileishmanial activity of EO fruit extract. Our results suggested that EO fruit extract could be used for the development of new phytomedicine against leishmaniasis.

Pyrrolidine-based 3-deoxysphingosylphosphorylcholine analogs as possible candidates against neglected tropical diseases (NTDs): identification of hit compounds towards development of potential treatment of Leishmania donovani.

A rational-based process was adopted for repurposing pyrrolidine-based 3-deoxysphingosylphosphorylcholine analogs bearing variable acyl chains, different stereochemical configuration and/or positional relationships. Structural features were highly influential on activity. Amongst, enantiomer 1e having 1,2-vicinal relationship for the -CH2O- and the N-acyl moieties, a saturated palmitoyl chain and an opposite stereochemical configuration to natural sphingolipids was the most potent hit compound against promastigotes showing IC50 value of 28.32 µM. The corresponding enantiomer 1a was 2-fold less potent showing a eudismic ratio of 0.54 in promastigotes. Compounds 1a and 1e inhibited the growth of amastigotes more potently relative to promastigotes. Amongst, enantiomer 1a as the more selective and safer. In silico docking study using a homology model of Leishmania donovani inositol phosphoceramide synthase (IPCS) provided plausible reasoning for the molecular factors underlying the found activity. Collectively, this study suggests compounds 1a and 1e as potential hit compounds for further development of new antileishmanial agents.

Boswellic Acids Show In Vitro Activity against Leishmania donovani.

In continuation of our search for leads from medicinal plants against protozoal pathogens, we detected antileishmanial activity in polar fractions of a dichloromethane extract from Boswellia serrata resin. 11-keto-ß-boswellic acid (KBA) could be isolated from these fractions and was tested in vitro against Leishmania donovani axenic amastigotes along with five further boswellic acid derivatives. 3-O-acetyl-11-keto-ß-boswellic acid (AKBA) showed the strongest activity with an IC50 value of 0.88 µM against axenic amastigotes but was inactive against intracellular amastigotes in murine macrophages.

Scaffold-Hopping Strategy on a Series of Proteasome Inhibitors Led to a Preclinical Candidate for the Treatment of Visceral Leishmaniasis.

There is an urgent need for new treatments for visceral leishmaniasis (VL), a parasitic infection which impacts heavily large areas of East Africa, Asia, and South America. We previously reported on the discovery of GSK3494245/DDD01305143 (1) as a preclinical candidate for VL and, herein, we report on the medicinal chemistry program that led to its identification. A hit from a phenotypic screen was optimized to give a compound with in vivo efficacy, which was hampered by poor solubility and genotoxicity. The work on the original scaffold failed to lead to developable compounds, so an extensive scaffold-hopping exercise involving medicinal chemistry design, in silico profiling, and subsequent synthesis was utilized, leading to the preclinical candidate. The compound was shown to act via proteasome inhibition, and we report on the modeling of different scaffolds into a cryo-EM structure and the impact this has on our understanding of the series' structure-activity relationships.

Bio-guided isolation of anti-leishmanial natural products from Diospyros gracilescens L. (Ebenaceae).

BACKGROUND: Plants represent an intricate and innovative source for the discovery of novel therapeutic remedies for the management of infectious diseases. The current study aimed at discovering new inhibitors of Leishmania spp., using anti-leishmanial activity-guided investigation approach of extracts from Diospyros gracilescens Gürke (1911) (Ebenaceae), targeting the extracellular (promastigotes) and intracellular (amastigotes) forms of Leishmania donovani. METHODS: The plant extracts were prepared by maceration using H20: EtOH (30:70, v/v) and further fractionated using a bio-guided approach. Different concentrations of D. gracilescens extracts, fractions and isolated compounds were tested in triplicate against L. donovani promastigotes and amastigotes in vitro. The antileishmanial potency and cytotoxicity on RAW 264.7 cells were determined using the resazurin colorimetric assay. The time kill kinetic profile of the most active sample was also investigated. The structures of all compounds were elucidated on the basis of extensive spectroscopic analyses, including 1D and 2D NMR, and HR-ESI-MS and by comparison of their data with those reported in the literature. RESULTS: The hydroethanolic crude extract of D. gracilescens trunk showed the most potent antileishmanial activity (IC50 = 5.84 µg/mL). Further fractionation of this extract led to four (4) fractions of which, the hexane fraction showed the most potent activity (IC50 = 0.79 µg/mL), and seven (07) compounds that exhibited moderate potency (IC50 = 13.69-241.71 µM) against L. donovani. Compound 1-deoxyinositol (7) inhibited the promastigote and amastigote forms of L. donovani with IC50 values of 241.71 µM and 120 µM respectively and also showed the highest selectivity against L. donovani promastigotes (SI > 5.04). To the best of our knowledge, the antileishmanial activity of this compound is being reported here for the first time. The promising hexane fraction showed significant inhibition of parasites growth at different concentrations, but with no evidence of cidal effect over an exposure period of 120 h. CONCLUSIONS: The results obtained indicated that the hydroethanolic extract from the D. gracilescens trunk and the derived hexane fraction have very potent inhibitory effect on cultivated promastigotes and amastigotes of L. donovani parasite. The isolated compounds showed a lesser extent of potency and selectivity. However, further structure-activity-relationship studies of 1-deoxyinositol could lead to more potent and selective hit derivatives of interest for detailed drug discovery program against visceral leishmaniasis.